Perforated Patch Clamp Recordings in ex vivo Brain Slices from Adult Mice

Intracellular signaling pathways directly and indirectly regulate neuronal activity. In cellular electrophysiological measurements with sharp electrodes or whole-cell patch clamp recordings, there is a great risk that these signaling pathways are disturbed, significantly altering the electrophysiological properties of the measured neurons. Perforated-patch clamp recordings circumvent this issue, allowing long-term electrophysiological recordings with minimized impairment of the intracellular milieu. Based on previous studies, we describe a superstition-free protocol that can be used to routinely perform perforated patch clamp recordings for current and voltage measurements.

Intracellular signaling pathways directly and indirectly regulate neuronal activity.In cellular electrophysiological measurements with sharp electrodes or whole-cell patch clamp recordings, there is a great risk that these signaling pathways are disturbed, significantly altering the electrophysiological properties of the measured neurons.Perforated-patch clamp recordings circumvent this issue, allowing long-term electrophysiological recordings with minimized impairment of the intracellular milieu.Based on previous studies, we describe a superstition-free protocol that can be used to routinely perform perforated patch clamp recordings for current and voltage measurements.

Background
Neuronal activity is controlled to a large extent by intracellular signaling systems.This study describes how to perform perforated-patch clamp recordings for high-quality single-electrode whole-cell current and voltage clamp recordings with minimized impact on the cytosolic integrity.Whole-cell patch clamp recordings have largely replaced intracellular recordings with sharp microelectrodes for single-electrode current and voltage clamp experiments.Because of the high seal resistance between the cell membrane and the recording electrode, this patch clamp configuration allows recording with a high signal-to-noise ratio, even from very small neurons.In addition, the whole-cell configuration provides low access resistance, which helps to ensure that the patch electrode solution can exchange freely with the cytoplasm.The whole-cell configuration is therefore also ideally suited to control the composition of the intracellular milieu, such as ion concentration, and to load the recorded neurons with tracers, sensors, and pharmacological agents.However, the free exchange of molecules between the cytoplasm and the patch electrode has a downside, as it impairs neuronal function by interfering with the cytosolic signaling system.Practically, this makes it impossible to use the wholecell recording configuration for long-term measurements without significantly altering the physiological state of the recorded neurons.The perforated-patch configuration, introduced initially by Lindau and Fernandez (1986) and Horn and Marty (1988), minimizes or even overcomes this drawback of the whole-cell configuration.Instead of rupturing the membrane under the recording electrode, which mediates the exchange between the cytosol and the electrode solution, pore-forming substances (ionophores) provide electrical access to the cell interior while largely maintaining the integrity of the cytoplasmic components of the neuron.The original and most used perforating agents have been the antibiotic polyenes nystatin, amphotericin B, and the antibiotic polypeptide gramicidin (Horn and Marty, 1988;Akaike, 1994;Akaike and Harata, 1994;Kyrozis and Reichling, 1995;Klöckener et al., 2011;Könner et al., 2011;Hess et al., 2013).While polyenes and the peptide exhibit differences in their pore-forming mechanisms and ion selectivity (Myers and Haydon, 1972;de Kruijff and Demel, 1974;Russell et al., 1977;Tajima et al., 1996), their pores share key common properties: they are permeable to small molecules with a molecular weight up to ~200 Da, including monovalent ions (Urry, 1971;de Kruijff et al., 1974;Kyrozis and Reichling, 1995).However, they are neither permeable to divalent ions like Ca 2+ nor intracellular signaling molecules of larger molecular weight.Building on previous studies, we describe here a protocol that can be used to routinely perform perforated-patch clamp recordings in brain slices of adult mice.The procedure is based on the use of amphotericin B as ionophore, as it has been, in our hands, the most suitable to achieve low access resistance and reproducibility.We describe how this approach can be used for current and voltage measurements and how this can be combined with single-cell labeling.We have applied this method to a variety of neuron types (see Table 1 for examples), but, for consistency, we only show data from substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons here.In the context of DA neuron recording, one reviewer has strongly suggested mentioning the work of Cattaneo et al. (2021) describing cell-attached and whole-cell patch clamp recordings from DA neurons in the SNpc.A defined volume of amphotericin B stock solution (see Table 1) is added to 1 mL of electrode solution, while the solution in the other microtube remains free of amphotericin (tip fill).In our experience, a final amphotericin concentration of 160-200 μg/mL (4-5 μL stock solution) ensures excellent perforation and recording conditions for many neuron types.However, depending on cell type and recording mode (current clamp or voltage clamp), optimizing the amphotericin concentration might be useful or even necessary (for examples, see Table 1).e. Shake (by hand) the amphotericin B-containing electrode solution several times.f.Add 1% (0.1 mg/100 μL) tetramethylrhodamine-dextran to the amphotericin B-containing electrode solution.Tetramethylrhodamine-dextran is used to check the integrity of the cell membrane once the ionophore has perforated the membrane (for further explanation, see below.)g.Put all three solutions (stock, tip fill, and amphotericin B-containing electrode solution) on ice.The amphotericin B-containing electrode solution can be used for up to three hours.After that, prepare a fresh solution.

C. Setting up the perforated-patch clamp recording for current clamp
1. Pull electrodes with resistances between 3 and 5 MΩ.
2. Fill the electrode tips with plain electrode solution (tip fill without amphotericin B).
3. For the backfill, use the amphotericin B-containing electrode solution.
4. Put the electrode in the bath but do not apply positive pressure!Note: Commonly, in patch clamp recordings from brain slices, positive pressure is applied when the electrode is advanced to the cell to prevent clogging of the electrode tip and to clean the target cell.However, in this case, the amphotericin B-containing solution would be driven into the electrode tip, severely compromising seal formation.5.Only when the electrode is positioned directly in front of the cell, apply a slight positive pressure with the mouthpiece and continue to approach the cell.As soon as a dent forms in the cell membrane in front of the electrode, immediately release the positive pressure (Figure 1C).6. Apply gentle negative pressure with the mouthpiece and constantly monitor the test pulse (voltage clamp mode, holding potential: 0 mV, test pulse amplitude: 5 mV) and the seal resistance.7. Release the pressure after you have reached a seal resistance of > 600 MΩ.
Note: Sometimes, you will not be able to reach a giga seal since the ionophore already starts perforating  The perforation process is reflected by an increase in action potential amplitude, indicating a decrease in series resistance.During this process, spontaneous conversions to the whole-cell configuration can occur, which become evident by an abrupt increase in action potential amplitude (Figure 2A).In many cases, this conversion is accompanied by hyperpolarization and the cessation of spiking activity (e.g., due to the opening of KATP channels).If no further changes in the parameters mentioned above occur, the perforation process is complete, and the actual measurements can begin.Note: To get meaningful measurements of electrophysiological properties of the investigated cell, the series resistance should ideally be well below 60 MΩ. 9.During the recording, check the tetramethylrhodamine-dextran fluorescence regularly to see if the membrane patch in the electrode is still intact (Figure 2B).If not, terminate the recording.Adding tetramethylrhodamine-dextran helps significantly to monitor the integrity of the perforated patch.This is very useful since spontaneous ruptures of the membrane patch are often not immediately reflected in noticeable changes in the action potential amplitude and the series resistance once the membrane has been perforated.Due to its molecular weight of 3,000 Da, tetramethylrhodamine-dextran can neither permeate through the cell membrane nor the pores formed by the ionophores, making it possible to detect whether the membrane patch has been ruptured.Note: In our experience, tetramethylrhodamine-dextran does not compromise the quality of the recording.
Although other fluorescent molecules may be suitable, checking whether they interfere with the recording is crucial.
Once the perforated configuration is established, long-term recordings can be performed with a significantly minimized rundown compared to the whole-cell configuration.Figures 3-5 show examples of perforated-patch clamp recordings in different experimental settings, illustrating the possibilities of this recording configuration, e.g., in long-lasting current and voltage clamp experiments.A direct comparison between the whole-cell and perforated configurations is shown in Figure 3.In the whole-cell configuration, action potential frequency decreased dramatically within the first 10 min (Figure 3A).In contrast, the frequency remained stable in the perforated-patch clamp recordings (Figure 3B).This configuration is, therefore, particularly suitable for long-term (> 2 h) pharmacological experiments where it is desirable to demonstrate reversibility and reproducibility in the same recording.Such an experiment is shown in Figure 4, where cocaine was bath-applied and washed out twice.

D. Setting up the perforated patch clamp recording for voltage clamp
To perform voltage clamp experiments, follow steps 1-7 of section C and then continue with the following steps.Note: Higher amphotericin B concentrations might be useful (see Table 1).
1. Switch the holding potential to -60 mV. 2. Continue applying 5 mV test pulses in voltage-clamp mode (Figure 1B). 3. Wait until the perforation has reached a steady state (stable current amplitude).4. Check the integrity of the membrane patch occasionally, as already described.
Examples of successful voltage clamp recordings are given in Figure 5.

Data analysis
Data were recorded with the PatchMaster software (HEKA).Data were sampled at 10 kHz and low-pass filtered at

12 .
Place the brain slices in the recording chamber for the recordings held down with a slice anchor and superfused with carbogenated ACSF at the desired experimental temperature.B. Preparation and usage of the ionophore (amphotericin B)1.Preparation a. Make a stock solution of amphotericin B by dissolving 4 mg of amphotericin B in 100 μL of DMSO.b.Sonicate the stock solution until the solution becomes a uniformly turbid yellowish solution.c.Vortex the stock solution several times.d.Prepare two 1.5 mL microtubes, each filled with 1 mL of electrode solution (for current or voltage clamp).

6 Published:
Cite as: Hess, S. (2023).Perforated Patch Clamp Recordings in ex vivo Brain Slices from Adult Mice.Bio-protocol 13(16): e4741.DOI: 10.21769/BioProtoc.4741.Aug 20, 2023 the cell membrane during the sealing process, which can take up to 20 min.8. Switch to current clamp and monitor the perforation process.Ideally, you should see a continuous transition from the on cell to the perforated configuration, as shown in Figure 1A.

Figure 1 .
Figure 1.Perforation process.Original recordings showing the transition from the on-cell to the perforated-patch recording configuration under current clamp (A) and voltage clamp (B).Bottom: segments of the original traces shown in the top panel in higher time resolution.The numbers indicate the times from which the segments originate.(B).Voltage pulses (5 mV, 5 ms, HP = -60 mV) were applied every 10 s.Bottom left: test pulse before perforation (RS > 100 MΩ), Bottom right: test pulse after perforation has reached a steady state (RS < 20 MΩ).HP, holding potential.(C).Seal formation.Top: slight positive pressure forms a dent in the cell membrane in front of the electrode.Bottom: after releasing the positive pressure, negative pressure supports seal formation.Scale bar: 10 μm.

Figure 2 . 7 Published:
Figure 2. Tetramethylrhodamine-dextran as a marker for rupture of the membrane.Spontaneous conversion from the perforated patch to the whole-cell configuration.(A).Current clamp recording with corresponding frequency plot showing the spontaneous rupture of the membrane patch during the

Figure 3 . 8 Published: Aug 20, 2023 Figure 4 .
Figure 3.Time course of spontaneous action potential frequency from 30 min measurements in whole-cell (A) and perforated-patch recordings (B).Whole-cell recordings (n = 9); perforated-patch recordings (n = 11).The traces at the bottom correspond to the red symbols in the respective frequency plots.

9 Published: Aug 20, 2023 Figure 5 . 10 Published: Aug 20, 2023 Figure 6 .
Figure 5. Perforated patch voltage clamp recordings of voltage-activated Ca 2+ currents (ICa).ICa was induced by depolarizing voltage steps to 0 mV (A) or -10 mV (B) from a holding potential of -60 mV every 10 s.A, B. Example of peak ICa in a mouse dopaminergic (DA) substantia nigra pars compacta (SNpc) neuron plotted over time during cadmium (A; 1 mM) or nifedipine (B; 10 μM) bath application.The numbers correspond to calcium current traces shown at the bottom.